1. Technical Field
The present invention relates to a process for quantitative and/or qualitative determination of the microbial contamination of aqueous suspensions, emulsions or dispersions containing minerals and/or pigments and/or fillers and/or fiber materials, optionally in combination with polymers in colloidal form.
2. Related Art
Determinations of the germ contamination and hygienic controls of aqueous suspensions by means of conventional methods essentially rely on the propagative capacity of the microorganisms to be detected. Naturally, the time required to perform these methods ranges from 24 h to several days. These time periods are too long for many questions, and the results are obtained too late to intervene in a guiding manner in production processes. Particularly performing controls prior to transport time and again requires methods which are characterized by short determination intervals.
Thus, the main problem of conventional microbiological analyses of aerobic mesophilic germs, such as Plate Count or Easicult, is their long incubation period of up to 48 h. By this methods it is impossible to obtain an evaluation and, thereby, determination of the germ count earlier than after the elapse of two days. In addition, several other effects must be considered such as the nutrient medium, partial pressure of oxygen (aerobic/anaerobic), selectivity, pH and much more.
Therefore, it is often impossible to determine the germ count of products, such as pigment slurries, prior to their shipment to customers and to intervene in a guiding manner by adding biocidal substances. Due to the long transport times required of up to 6 weeks by deep sea ship or rail, the pigment slurry may deteriorate or become unusable. The white pigment slurry may develop a gray color and start to smell. To date, preventive over-dosage of biocidal substances in the pigment slurry is the only possibility to exclude spoilage, is very cost-intensive, dissipates resources, and is ecologically nonsensical. Moreover, two different approaches are necessary to analyze for both aerobic mesophilic germs and fungi. Furthermore, the preparation of serial dilutions is necessary, thus multiplying the number of analyses.
Conventional methods for the determination of germs in the paper and pigment industries have been for example described in the xe2x80x9cSchweizerisches Lebensmittelbuchxe2x80x9d, chapter 56, section 7.01, 1985 edition, 1988 revised version, xe2x80x9cBestimmung von aeroben Bakterien und Keimenxe2x80x9d, and in the xe2x80x9cSchweizerisches Lebensmittelbuchxe2x80x9d, chapter 56, section 7.22, 1985 edition, 1988 revised version, xe2x80x9cBestimmung von Pilzenxe2x80x9d. Generally, prior to performing a determination in each case an incubation period of about 48 hours is required.
The CellFacts(copyright) particle analyzer and method have been developed by Microbial Systems company, Ltd. More detailed information may be obtained from Labor flash 9/96, Zeitung mit Leserdienst fxc3xcr Labor und Forschung, Ott Verlag+Druck AG, Ch-3607 Thun, Switzerland. Thus, it has been mentioned that the CellFacts analyzer may also be used to carry out determinations of the germ count in calcium carbonate slurries. However, experiments performed in the applicant""s laboratory revealed that a determination of this type in the manner described by the manufacturer is impossible or only inaccurate.
The principle of the measurement performed by the CellFacts analyzer is based on the measurement of bacteria, fungi, and yeasts in the form of particles in an electrical field wherein the number of particles is determined by an interval incubation of the samples and subsequent measurement of the increase in particle count and, in the case of exponential growth, by extrapolation to t0. This measurement principle is also effective in measuring a xe2x80x9clowxe2x80x9d number of xe2x80x9cforeign particlesxe2x80x9d having the same or a similar size as bacteria, fungi, and yeasts. In the case of suspensions and emulsions containing a proportion of xe2x80x9cforeign particlesxe2x80x9d, such as minerals, fillers and/or pigments, of  greater than 1% by wt. the number of inert xe2x80x9cforeign particlesxe2x80x9d present having the same size as microorganisms, namely 0.5-20 xcexcm, i.e. the blank value t0, is too high to enable the detection of a further increase of the germs by propagation in the incubator within a period of  less than 10 hours. Thus, the CellFacts analyzer is unable to perform a sufficiently exact measurement, and the manufacturer requires a dilution of the starting liquid to ensure applicability.
At a blank value of 108 particles/ml, it is impossible to significantly detect an increase of 103 particles/ml by means of the CellFacts analyzer. However, the dilution required due to the presence of the xe2x80x9cforeign particlesxe2x80x9d is too high so that the dilution of the reproductive organisms which of course are diluted by the same order of magnitude is no longer significant, and the result obtained is incorrect. Furthermore, xe2x80x9cforeign particlesxe2x80x9d having a diameter of  greater than 20 xcexcm may plug the measurement cell which has a diameter of only 30 xcexcm. The xe2x80x9cforeign particlesxe2x80x9d may for example be of mineral type, such as calcium carbonate, synthetic, organic, of polymeric type, such as polystyrene acrylate dispersions, or of natural, organic type, such as starch solutions or hemicelluloses and/or cellulose fibers, or a combination of the above particles as they are for example present in a paper mill cycle.
It is an object of the present invention to provide a quick and powerful, easily performed process for the quantitative and/or qualitative determination of the microbial contamination of aqueous suspensions, emulsions or dispersions containing minerals and/or pigments and/or fillers and/or fiber materials wherein said process avoids the above described disadvantages of the prior art.
According to the present invention, this object has been solved by a process according to the generic part of claim 1 characterized in that a sample of the suspensions, emulsions or dispersions, following the addition of one or more organic substances which can be degraded by microorganisms and optionally following a subsequent incubation, is centrifuged to separate the microorganisms from the minerals and/or fillers and/or pigments and/or fiber materials and the number and/or size and/or nature of the microorganisms in the aqueous supernatant phase is determined after one or more incubations.
Preferred embodiments of the present invention will become obvious from the dependent Claims as well as the following Specification and the Examples.
Since several years, the quantitative and qualitative determination of microorganisms belongs to the prior art. However, the determination of microbial contamination is particularly difficult in cases where a high concentration of other solid particles such as minerals, pigments, fillers and/or fiber materials is present in the sample to be investigated. Because these xe2x80x9cforeign particlesxe2x80x9d often have a size similar to microorganisms, generally, it is only possible to minimize the number of such xe2x80x9cforeign particlesxe2x80x9d by carrying out high dilutions. However, concomitantly and unavoidably, these high dilution steps reduce the number of contaminating microorganisms, and a long incubation period is required to increase the number of microorganisms by means of propagation to an extent that enables safe detection.
Therefore, there has been a need to create a new process of the type mentioned in the beginning which provides reproducible and reliable results with respect to the number, size and/or type of the microorganisms in the sample to be investigated in a substantially shorter time.
Surprisingly and unexpectedly, it has been discovered that it is possible to separate the microorganisms from inert materials (xe2x80x9cforeign particlesxe2x80x9d) by addition of degradable organic substances to the sample and performing a subsequent centrifugation. Usually, the microorganisms preferably adhere to the surface of the mineral, pigment, filler and/or fiber materials making it difficult to separate them from the surface. Although the xe2x80x9cforeign particlesxe2x80x9d are sedimented by a simple centrifugation step, however, the microorganisms are drawn along into the pellet because they preferably adhere to those particles, and thereby the portion of microorganisms remaining in the supernatant provides incorrect values with respect to the degree of contamination with microorganisms.
It has been shown that the addition of biologically degradable organic substances surprisingly enables a separation of the microorganisms and the minerals, pigments, fillers and/or fiber materials. Preferably, the organic substance which can be degraded by microorganisms is a nutrient medium conventionally used in the cultivation of microorganisms. Surprisingly, this nutrient medium acts as a separation agent between the microorganisms and the foreign particles and, thus, for the first time enables the separation of and distinction between the two without affecting further steps of the microorganism analysis. Substances which may not be biologically degraded or whose biological degradation is difficult bear the risk that the microorganisms are separated from the inert particles while at the same time the substances act as inhibitors of microorganism growth and thus lead to inaccurate results.
In the process according to the present invention not only one process step, namely the dilution step, may be omitted but in addition the incubation period is extraordinarily reduced. Furthermore, there is not just added a separation agent but the separation agent simultaneously acts as a nutrient solution which may be employed for optimal propagation of the microorganisms to be investigated and, in a preferred embodiment, is selected to be specific for a particular microorganism type to be tested, such as bacteria, fungi or yeasts.
It was only by the addition of an organic substance which may be degraded by the microorganisms, preferably in the form of a nutrient solution or medium, respectively, and by introduction of a centrifugation step that the object according to the present invention has been solved. A reduction in analysis time or even the possibility to perform an analysis at all has been achieved because the sample to be tested contains a smaller number of xe2x80x9cforeign particlesxe2x80x9d. Moreover, it is possible to use a smaller starting number and a smaller increase in microbiological particles over time than with samples having a higher blank value, i.e. a higher number of xe2x80x9cforeign particlesxe2x80x9d.
By the process according to the invention, aqueous suspensions, emulsions and dispersions may be investigated which among other substances contain minerals, pigments, fillers and/or fiber materials, such as cellulose fibers, and which optionally further contain polymers such as natural, synthetic or semisynthetic polymers in colloidal form. Examples of such polymers are: styrene butadiene, styrene acrylate, melamine resins, formaldehyde urea resins, starch, carboxymethylcellulose.
Preferably, the samples to be tested are derived from the paper processing industry. Further areas of use are the pigment industry and the metal processing industry.
According to the present invention, a sample of the suspensions, emulsions or dispersions to be tested for microbial contamination is taken. Generally, the amount of the sample obtained is 0.5-20 ml, however, a smaller or higher amount may be taken. Besides, the amount of sample taken is not important for the success of the process of the invention.
The sample obtained is added and mixed with organic substances which may be degraded by microorganisms. The amount of biodegradable organic substances added to the sample is 0.5-50 ml of biodegradable substance per ml of sample.
The ratio of the sample volume to the volume of biodegradable substance is dependent on the original concentration of the sample and the concentration of biodegradable substance in solution. The ratio is adjusted to an amount of biodegradable substance which is large enough to enable a separation of the microorganisms and the minerals, fillers, pigments and/or fiber materials and optionally also polymers in colloidal form. The optimal ratio of the sample volume to the volume of biodegradable substance which must be determined in each case may be determined by the skilled artisan by means of manual experimentation.
Usually the concentration of the solution containing the biodegradable substance is selected to achieve a ratio of the sample solution to the solution containing the biodegradable substance of 1:0.1 to 1:100. The optimal ratio is strongly dependent on the concentration and the composition of the respective sample solution. Pigment and/or filler suspensions having a solids content of  greater than 65% by wt. are generally diluted in a ratio of 1:2 to 1:10, preferably in a ratio of 1:3. If the solids content is  less than 65% by wt., the dilution is usually performed in a ratio of 1:0.1 to 1:1. In certain cases, especially if a very high contamination can be expected, the dilution is generally performed in a ratio of 1:10 to 1:100. The chosen dilution factor must be considered in the subsequent evaluation of the contamination or must be pointed out specifically in the result.
The biodegradable organic substances particularly include the group of nutrient media which are specific for a particular species of microorganism to be tested. Preferably, nutrient media contain a source of carbon, nitrogen, phosphate and/or sulfur as well as optionally minerals, growth factors and/or vitamins. Other substances may be added to the nutrient medium if they are required for optimal growth of the microorganisms. In the following, there are described preferred media which may be employed according to the present invention.
Optionally, following addition of the nutrient medium one or more incubation steps may be carried out to enhance the number of microorganisms in the sample. This may be especially advantageous for a subsequent qualitative analysis. In a preferred embodiment of the invention, however, this incubation prior to centrifugation is omitted leading to a marked time reduction.
To separate the microorganisms from the minerals, fillers, pigments and/or fiber materials a centrifugation step is carried out following the addition of the nutrient medium and the optional subsequent incubation step. The centrifugation step is performed in a manner to remove most of the microorganisms, i.e. more than 50%, from the xe2x80x9cforeign particlesxe2x80x9d, i.e. the minerals, fillers, pigments, polymers, and fiber materials. The centrifugation must be effective to accumulate the microorganisms in the upper phase while the foreign particles are sedimented. For this purpose, the centrifugation is preferably carried out at 100 to 1500 g, preferably 200 to 1200 g and particularly preferred at 600 to 1000 g. The gravity field is adjusted depending on the minerals, fillers, pigments, and fiber materials to be separated or depending on the microorganisms to be separated, respectively.
The optimal sedimentation index may be determined by the skilled artisan by means of experimentation. The centrifugation itself is performed for a time of 1 to 30 minutes, preferably 2 to 15 minutes and particularly preferred 5 to 10 minutes. The optimal centrifugation time may be determined by the skilled artisan performing laboratory experiments.
As the minerals and/or fillers and/or pigments there are preferably used: compounds containing elements of the second and/or third main group and/or fourth main group and/or fourth side group of the periodic system of the elements, particularly calcium and/or silicon and/or aluminium and/or titanium and/or barium and/or organic pigments.
As the minerals, fillers, and pigments, there are preferably used minerals and/or fillers and/or pigments containing kaolin and/or aluminium hydroxide and/or titanium hydroxide and/or barium sulfate and/or polystyrene hollow spheres and/or formaldehyde resins and/or calcium carbonate, particularly natural calcium carbonates and/or marble and/or lime and/or dolomite and/or calcium carbonates containing dolomite and/or synthetically prepared calcium carbonates, so-called precipitated calcium carbonated.
The supernatant containing the microorganisms is removed and may then be used directly in a quantitative and/or qualitative determination of the microbial contamination. Preferably, this is followed by one or more incubation steps to increase the number of microorganisms in the supernatant. This propagation step is performed at an incubation temperature which favors the microorganisms and is dependent on the type of microorganisms to be propagated. Preferred incubation temperatures are in the range of 20 to 37xc2x0 C., further preferred 28 to 34xc2x0 C., and particularly preferred 31.5 to 32.5xc2x0 C. The incubation temperatures which are necessary in each case are known to the skilled artisan and may be either found in monographs or may be experimentally determined.
The total time of the individual incubation steps at an incubation temperature of 30xc2x0 C. is up to 12 hours at a contamination of less than 105 germs/ml of sample, up to 6 hours at a contamination of more than 105 germs/ml sample but less than 106 germs/g of sample, and up to 3 hours in a suspension with an original solids content of 60-80% by wt. and a contamination of more than 105 germs/ml (using tryptic soy broth agar).
The sum of the individual incubation steps at 30xc2x0 C. is up to 2 to 6 at a contamination of less than 105 germs/ml of sample, 2 to 4 at a contamination of more than 105 germs/ml sample but less than 106 germs/g of sample, and 2 to 3 in a suspension with an original solids content of 60-80% by wt. and a contamination of more than 105 germs/ml (using tryptic soy broth agar).
The individual incubation times at an incubation temperature of 30xc2x0 C. are 1 to 12 hours at a contamination of less than 105 germs/ml of sample, 1 to 6 hours at a contamination of more than 105 germs/ml sample but less than 106 germs/g of sample, and 1 to 2 hours in a suspension with an original solids content of 60-80% by wt. and a contamination of more than 105 germs/ml (using tryptic soy broth agar).
Several methods known from the prior art are available for the quantitative or qualitative determination, respectively, of the microorganisms thus obtained. Particularly preferred according to the present invention is the CellFacts(copyright) analysis or the ATP method. Other methods are available and are known to those skilled in the art. The preferred methods are described in more detail in the following Examples.
After the process according to the present invention has been carried out, a qualitative and/or quantitative determination of the microorganisms may be performed. First, qualitative determination means a rough differentiation between the groups of fungi, yeasts and bacteria. If the CellFacts(copyright) analysis is calibrated which specific microorganisms such as specific bacteria also a further specification within individual subtypes may be performed. The qualitative analysis, for example via the CellFacts(copyright) method, is performed on the basis of a gross differentiation with respect to size or volume, respectively, of a single cell.
The type of the nutrient media employed as the separation agent particularly depends on the microorganism to be separated or propagated. Preferably, nutrient media are employed which enable the selective growth of the germs to be determined and, if possible, suppress the growth of other germs not to be determined. Examples of nutrient media which may be employed according to the present invention are tryptone azolectin/Tween 20 (TAT) broth agar, glucose solution, peptone/casein nutrient broth, preferably tryptic soy broth agar. The composition of the nutrient media is given in annex to this specification.
The concentration of the nutrient medium for the microorganisms is preferably in the range of 0.1 to 10% by wt., more advantageously 2 to 5% by wt., and particularly advantageous about 3% by wt.
The process according to the present invention not only enables a qualitative but also a quantitative determination of the bacteria, fungi and yeasts. In a preferred embodiment, this analysis is performed in one step.
Following the separation of microorganisms and minerals, fillers, pigments and/or fiber materials, i.e. the xe2x80x9cforeign particlesxe2x80x9d, one or more, preferably two to five incubation steps are carried out to increase the number of microorganisms present in the sample supernatant.
Multiple incubations are performed in intervals, i.e. the measurement of the contamination is in each case repeated after the time t0+x. This enables the limitation of the incubation time and the incubation intervals. At the time when exponential growth is observed an extrapolation and a calculation back to the time t0 is performed. Thus, a high original degree of contamination will result in a growth which can be observed and calculated earlier so that subsequent incubation intervals may be omitted.
The incubation time at a contamination of less than 105 germs/ml of sample is up to 12 hours, at a contamination of less than 106 germs/ml up to 6 hours, in a suspension originally containing 60-80% by wt. of solids and having a contamination of more than 105 germs/ml sample up to 3 hours wherein in the last case the use of tryptic soy broth agar is preferred. The incubation time which is to be used depending on the degree of contamination with microorganisms may be determined by those skilled in the art by simple manual experimentation. The lower the original degree of microbial contamination in the sample the longer are the incubation times to be selected and vice versa. The original portion of solids in suspension does not directly affect the incubation time.